The purpose of the project is to study the role of iron availability on the pathogenesis of Actinobacillus actinomycetemcomitans (A. a.). Little is known about the iron transport systems of anaerobic bacteria, although it is clear that the acquisition of iron from the host is required in order to sustain growth. The identification of an iron regulated membrane protein of 70kD that is antigenic in humans may represent a vaccine candidate for the prevention and treatment of A.a. infections. The specific aims of the study are to identify potential in vivo iron sources utilized by A.a. to help in targeting iron transport systems. In addition, the role of the 70kD iron regulated membrane protein in iron uptake will be studied. To achieve these goals an iron limited growth medium will be developed in order to study iron utilization with a limited number of variables. Separation of the 70kD protein from other membrane proteins will be achieved with sodium dodecyl sulfate gel electrophoresis and the protein will be subsequently transferred to a PVDF membrane. The 70kD band will be excised and sequenced using an automated gas-phase sequencer. The derived N-terminal amino acid sequence will be utilized to generate an oligonucleotide probe specific for the 70kD protein and used for screening a lambama gt11 library of A.a. DNA. As an additional strategy, monospecific antisera will also be utilized to screen the library. Once the wild-type gene has been identified, it will be cloned into an appropriate vector and utilized to generate mutations in the wild type gene in order to characterize the function of the protein in iron uptake.